Tumor necrosis factor .alpha. (TNF.alpha., also cachectin), discovered as a result of its haemorragic-necrotizing activity on certain tumors, and lymphotoxin (TNF.beta.) are two closely related peptide factors [3] from the class of lymphokines/cytokines which are both referred to hereinafter as TNF [see references 2 and 3]. TNF possesses a broad cellular spectrum of activity. For example, TNF has inhibitory or cytotoxic activity on a series of tumor cell lines [2, 3], stimulates the proliferation of fibroblasts and the phagocytic/cytotoxic activity of myeloic cells [4, 5, 6], induces adhesion molecules in endothelial cells or exerts an inhibitory activity on the endothelium [7, 8, 9, 10], inhibits the synthesis of specific enzymes in adipocytes [11] and induces the expression of histo-compatibility antigens [12]. Many of these TNF activities are produced via induction of other factors or by synergistic effects with other factors such as interferons or interleukins [13-16].
TNF is involved in pathological conditions such as shock states in meningococcal sepsis [17], the development of autoimmune glomerulonephritis in mice [18] and cerebral malaria in mice [19] and human beings [41]. The toxic effects of endotoxin appear to be mediated by TNF [20]. Furthermore, TNF can trigger interleukin-1 fever [39]. On the basis of its pleiotropic functional properties, TNF in interaction with other cytokines is involved in additional pathological conditions as a mediator of immune response, inflammation, and other processes.
These biological effects are mediated by TNF via specific receptors. According to present knowledge not only TNF.alpha., but also TNF.beta. bind to the same receptors [21]. Different cell types differ in their number of TNF receptors [22, 23, 24]. Generally known TNF-binding proteins (TNF-BP) have been detected by covalent bonding to radioactively labelled TNF [24-29], and the following apparent molecular weights of the TNF/TNF-BP complexes obtained have been determined to be: 95/100 kD and 75 kD [24], 95 kD and 75 kD [25], 138 kD, 90 kD, 75 kD and 54 kD [26], 100.+-.5 kD [27], 97 kD and 70 kD [28] and 145 kD [29]. One such TNF/TNF-BP complex was isolated by anti-TNF-antibody immune affinity chromatography and preparative SDS-polyacrylamide gel electrophoreses (SDS-PAGE) [27]. The reductive cleavage of this complex and subsequent SDS-PAGE analysis gave several bands which were not tested for TNF-binding activity. Since the specific conditions which must be used for the cleavage of the complex lead to inactivation of the binding protein [31], the latter has also not been possible. The separation of soluble TNF-BP from human serum or urine by ion exchange chromatography and gel filtration (molecular weight in the region of 50 kD) was described by Olsson et al. [30].
Brockhaus et al. [32] obtained an enriched TNF-BP preparation from membrane extracts of HL.sub.60 cells by TNF.alpha.-ligand affinity chromatography and HPLC which, in turn, was used as an antigen preparation for the production of monoclonal antibodies against TNF-BP. Using such an immobilized antibody (immune affinity chromatography) Loetscher and Brockhaus obtained an enriched preparation of TNF-BP [31] from an extract of human placenta using TNF.alpha.-ligand affinity chromatography and HPLC, which gave a strong broad band at 35 kD, a weak band at about 40 kD and a very weak band in the region between 55 kD and 60 kD on SDS-PAGE analysis. Moreover, the gel showed a protein background smear in the region of 33 kD to 40 kD. The significance of these protein bands was, however, not clear due to the heterogenicity of the starting material which was used (placenta tissue; combined material from several placentas). In the state of the art TNF-BP have already been characterized by a N-terminal partial sequence [European Patent Application, Publication No. 308 378], whereby this sequence differs from the N-terminal partial sequence according to formula (IA) in accordance with the invention. Moreover, the TNF-binding proteins described in the state of the art are soluble, i.e. non-membrane bound, TNF-BP and not membrane-bound, i.e. insoluble, TNF-BP isolated from urine.